The long range goal of this project is to understand at the molecular level the basis for the exchange and maintenance of genetic information in Escherichia coli. This organism provides and excellent model system for the analysis of these complex processes because of its ease of manipulation and the wealth of genetic information delineating the existence of multiple pathways for genetic recombination and DNA repair. In addition, there is a growing body of data that suggests the existence of similar systems in a variety of eukaryotic organisms. To help achieve our objectives, the experiments outlined in this proposal will focus on three enzymes that have been implicated in genetic recombination, DNA repair and spontaneous mutagenesis. These proteins are exonuclease V/RecBCD enzyme (recB recC recD), DNA helicase II (uvrD) and exonuclease I (sbcB/xonA). Specific questions to be addressed include: 1. Isolation and characterization of mutations in the ATP binding sites of both the RecB and RecD subunits of exonuclease V. 2. Examination of the interactions between the RecB, RecC and RecD subunits of exonuclease V by isolating and characterizing mutations that suppress either recB or recC alleles but map in a different subunit of the enzyme. 3. Determine the actual function of DNA helicase II in mismatch repair versus excision repair through the characterization of a variety of mutant DNA helicase II proteins. 4. Ascertain why rep uvrD double mutants are inviable through the use of temperature sensitive uvrD mutations. 5. Determine what distinguishes sbcB from xonA alleles in the suppression of recB and recC mutations by purifying and characterizing the sbcB15 encoded exonuclease I.
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